The present invention relates to improvements in magnetic separation methodology, having particular utility in various laboratory and clinical procedures involving biospecific affinity reactions. Such reactions are commonly employed in testing biological samples for the determination of a wide range of target substances, including antigens, specific antibodies, specific biological factors such as vitamins, cell subpopulations (both eucaryotic and procaryotic), cell components, viruses and specific nucleic acid sequences, as in the case of gene probe analysis.
As used herein, the term "target substance" refers to any member of a specific binding pair, i.e., a pair of substances exhibiting a mutual affinity of interaction, and includes biospecific ligands and receptors. "Ligand" is used herein to refer to substances, such as antigens, haptens and various cell-associated structures, having at least one characteristic determinant or epitope which is capable of being biospecifically recognized by and bound to a receptor. "Receptor" is used herein to refer to any substance or group of substances having a biospecific binding affinity for a given ligand, to the exclusion of other substances. Among the receptors determinable via biospecific affinity reactions are antibodies (both polyclonal and monclonal), antibody fragments, and nucleic acids. The determination of any member of a biospecific binding pair is dependent upon its selective interaction with the other member of the pair.
The term "antigen", as used herein, refers to any substance to which antibodies can be produced, and includes haptens which can be made immunogenic by means known to those skilled in the art. The term "specific antibodies" is used herein to refer to antibodies produced in response to a specific stimulation of the host, be that stimulation due to specific exogenous agents, e.g., germ entities or specific inoculation, or stimulation resulting from endogenous components. Many biologically relevant target substances other than antigens and specific antibodies can be recognized via biospecific affinity reactions, e.g., the members of specific binding pairs such as Vitamin B-12/intrinsic factor. "Gene probes" include all pairs of complementary nucleic acid sequences which can undergo stable hybridization reactions, such pairs resulting from naturally occurring complementary base pair reactions or from reactions involving appropriately synthesized complementary oligonucleotides. Cells, e.g. erythrocytes or leukocytes, microorganisims, e.g. bacteria, and viruses are also separable from a mixed population thereof, by reason of their interaction with specific receptors, in accordance with the present invention.
Various methods are available for determining all of the above-mentioned target substances based upon complex formation between the substance of interest and its specific binding partner. Means are provided in each instance whereby the level of target substance/binding partner complex formation is detectable.
In the case of a competitive immunoassay to determine antigen, for example, the antigen of interest in a test sample competes with a known quantity of labelled antigen for a limited quantity of specific antibody binding sites. Thus, after an appropriate reaction period the amount of labeled antigen bound to specific antibody is inversely proportional to the quantity of antigen in the test sample. Competitive assays for antibodies, employing labeled antibodies (typically monoclonal antibodies) rather than labeled antigen, function in an analogous manner. In contrast, immunometric assays, commonly known as "sandwich" assays, involve the formation of a sandwich whose "layers" are: antibody/multivalent (minimally bivalent) antigen/antibody. When one of the antibodies is labeled, the amount of labeled antibody which is bound for each complete sandwich complex (antibody/antigen/antibody) is directly proportional to the amount of target antigenic substance present in the test sample. Sandwich assays can be performed in multi-step fashion with polyclonal antibodies or in fewer steps when monoclonals directed to independent antigenic determinants are employed. In both of the foregoing immunoassay techniques quantitation requires a physical separation of bound from free labeled ligand or labeled receptor. Such assays are known as heterogeneous assays.
In the case of non-antigen/antibody biospecific binding pairs, such as the determination of Vitamin B12 with its biospecific binding partner, intrinsic factor, a test sample being assayed for the presence of the vitamin is contacted with a known amount of the same vitamin bearing a suitable label and a limited quantity of intrinsic factor. Cobalt is commonly used as the label in such assays. Just as in the case of the competitive immunoassay technique described above, the amount of label bound to intrinsic factor is inversely proportional to the amount of vitamin in the sample. In such an assay bound/free separation must be performed for purposes of quantitation.
For gene probe analysis, a competitive assay format may be employed which is analogous to the above described competitive immunoassay techniques. A sandwich type assay, such as that described above, may also be employed. Again, separation of bound label from unbound label in the test sample is required for quantitation.
The same basic approach involving bound/free separation is routinely used in cell separation techniques.
Various methods have been reported for performing bound/free separations in heterogeneous systems in order to determine the bound/free ratio of labeled ligand or receptor. These may be classified for purposes of this discussion into liquid phase and solid phase techniques. Liquid phase separation techniques include (1) adsorption of free ligand onto materials which may be filtered or centrifuged, such as cellulose powder, dextan-coated charcoal, Kaolin, and the like, where a differential adsorption of ligand vs. receptor is possible; (2) non-specific or specific precipitation of antibody or receptor; (3) chromatographic separation based on molecular size; and (4) electrophoresis based on mobilities of components of the test sample; see, for example, Methods of Investigative and Diagnostic Endocrinology, Part I, pp. 84-120, S. Berson et al. eds., American Elsevier, N.Y. (1973).
One of the liquid phase separation techniques commonly employed involves the use of second antibodies. As an example, a competitive immunoassay may be performed by employing rabbit antibodies reacting with an appropriately labeled antigenic substance corresponding to the target substance of interest. After an appropriate incubation period, labeled antigen bound specifically to the rabbit antibodies can be determined by removing the rabbit antibodies from solution. Antibody removal can be achieved by forming a specific precipitate using second antibodies that immunospecifically interact with the rabbit antibodies. Thus, by adding an appropriate quantity of goat anti-rabbit IgG, for example, the rabbit IgG antibodies in the fluid phase are removed from solution and bring with them specifically bound labeled antigen. With appropriate washing of the resultant precipitate the quantity of labeled antigen bound to the rabbit antibodies can be quantitatively determined.
The Farr method, involving ammonium sulphate ((NH.sub.4).sub.2 SO.sub.4) precipitation of low molecular weight, hapten-like, labeled antigens, typifies the non-specific liquid phase separation methodology. R. Farr, J. Infect. Dis., 103:239 (1958). The Farr method is performed by adding a sufficient quantity of ammonium sulphate to the reacted test sample to cause precipitation of the bound antibodies therein, leaving behind the unbound labeled antigen. In employing the Farr method, as well as certain other liquid phase separation techniques, e.g., those involving precipitation with alcohol, dioxane and polyethylene glycol, it is important that the labeled antigen be unaffected by ammonium sulphate, or other protein precipitation agent.
The above-mentioned second antibody technique has been improved by the addition of promoters, e.g., polyethylene glycol, to the test sample to promote precipitation of lesser quantities of specific antibody/second antibody precipitates. The crosslinking of antibodies to facilitate phase separation can be done either before or after equilibration with the labeled antigen. In the former case, the cross-linked or aggregated antibody is a convenient reagent. Reagent so used ceases to fit the liquid phase separation classification, however, as not all reagents are in solution at the start of the assay.
The foregoing assays employing liquid phase separation techniques involve transforming a soluble biospecific binding partner to a precipitate that results either from crosslinking via non-covalent bonding or from the conversion of macromolecules, which exist in a colloidal state, to an insoluble form. Such conversions via "salting out" or the use of "non-solvent" proceed via well known colloidal properties of the macromolecules involved. See, A. Alexander et al., Colloid Science, Volumes 1 and 2, Oxford Press, London, (1949).
More recently, assays involving biospecific binding pairs have utilized solid phase separation techniques. Probably the simplest approach in solid phase separation consists of coating the interior surface of a suitable vessel with one member of the biospecific binding pair of interest, thus creating a stationary solid phase device. Methods for absorption or covalent attachment of the required reagents to such surfaces are well known to those skilled in the art. Solid supports may also comprise surfaces which can be emersed into a test medium. Phase separation simply entails removing the medium from contact with the surface after sufficient time has elapsed for biospecific interaction to occur between the binding pair members.
Among the more commonly used solid phase separation techniques are those employing finely divided inert particles (charcoal, derrivatized glass, cross-linked carbohydrate and/or polymer particles, as well as beads, e.g. latex), or even cross-linked insolubilized antibodies. Such particles or beads may be used to adsorb unbound materials directly, or through appropriate processing, may be used to create a solid phase bearing the appropriate binding partner. Bound/free separations may be accomplished gravitationally, e.g., by settling, or, alternatively, by centrifugation of the finely divided particles or beads from solution. If desired, such particles or beads may be made magnetic to facilitate the bound/free separation step. Magnetic particles are well known in the art, as is their use in immuno-and other biospecific affinity reactions. See, U.S. Pat. No. 4,554,088 and Immunoassays for Clinical Chemistry, pp. 147-162, Hunter et al. eds., Churchill Livingston, Edinborough (1983). Generally, any material which facilitates magnetic or gravitational separation may be employed as a solid support. Solid phase materials which are particulate and free to move in solution, such as those described above, are known as mobile solid phase reagents.
Small magnetic particles have proved to be quite satisfactory as a mobile solid-phase, as they provide very high surface areas, give reasonable reaction kinetics and can readily be removed from solution by means of commercially available magnetic devices (Ciba-Corning Medical Diagnostics, Wampole, Mass.; Serono Diagnostics, Norwell, Mass.). Magnetic particles ranging from 0.7-1.5 microns have been described in the patent literature, including, by way of example, U.S. Pat. Nos. 3,970,518; 4,018,886; 4,230,685; 4,267,234; 4,452,773; 4,554,088; and 4,659,678. Several of these particles are useful solid supports, having reasonably good suspension characteristics when mildly agitated. Insofar as is known, however, absent some degree of agitation, all of the magnetic particles presently in commercial use settle in time and must be resuspended prior to use. This adds another step to any process employing such reagents.
Small magnetic particles, such as those mentioned above, generally fall into two broad categories. The first category includes particles that are permanently magnetized; and the second comprises particles that become magnetic only when subjected to a magnetic field. The latter are referred to herein as magnetically responsive particles. Materials displaying magnetically responsive behavior are sometimes described as superparamagnetic. However, certain ferromagnetic materials, e.g., magnetic iron oxide, fall into the magnetically responsive category when the crystal is about 300 A or less in diameter. Larger crystals of ferromagnetic materials, by contrast, retain permanent magnet characteristics after exposure to a magnetic field and tend to aggregate thereafter. See P. Robinson et al., Biotech Bioeng. XV:603-06 (1973).
Magnetically responsive colloidal magnetite is known. See U.S. Pat. No. 4,452,773 and published International Application PCT WO 87/02063. Magnetically responsive materials that behave as true colloids are characterized by their stability to gravitational separation from solution for extended periods of time and their sub-micron particle size, which is generally less than about 200 nanometers (0.20 microns). Such materials are believed to be composed of a crystalline core of superparamagnetic material surrounded by molecules, which may be physically absorbed or covalently attached, and which confer stabilizing colloidal properties. It is further believed that such colloidal materials are so small that they do not contain a complete magnetic domain and that their Brownian energy exceeds their magnetic moment. As a consequence, North Pole, South Pole alignment and subsequent mutual attraction/repulsion of these colloidal materials does not appear to occur even in moderately strong magnetic fields, contributing to their solution stability. Accordingly, colloidal magnetic materials are not readily separable from solution as such, even with powerful electromagnetics but, instead, require gradient field separation techniques. See, R. R. Oder, IEEE Trans. Magnetics, 12:428-35 (1976); C. Owen and P. Liberti, Cell Separation: Methods and Selected Applications, Vol. 5, Pretlow and Pretlow eds., Academic Press, N.Y., (1986). The gradient fields normally used to filter such materials generate huge magnetic forces.
Solid phase systems in which a member of a specific binding pair, or a reagent which will bind one member of the pair, is immobilized not only facilitate phase separation, but permit separation to be accomplished simply and rapidly. See, for example, U.S. Pat. No. 4,271,140. The speed with which such separations can be performed is vital in the case of biospecific affinity reactions, as significant re-equilibration of the biospecific binding pair members may take place given sufficient time. Theoretically, antibody interacting with low molecular weight labeled antigen and test antigen could be separated from solution by ultracentrifugation but the time factor and difficulty of such a process would be impractical. For similar reasons, electrophoretic and chromatographic means for such separations are not often used.
The aforementioned U.S. Pat. Nos. 3,970,518 and 4,018,886 to Giaever relate to biological separations using protein-coated small magnetic particles. These patents describe an apparatus and separation method utilizing magnetic particles, ranging in size from colloidal to 10 microns, coated directly with antibody that interacts specifically with the biological entities of interest. However, the above-noted U.S. Pat. No. 4,230,685 to Sengei et al., which discloses the preparation of magnetic particles and their use in cell and other separations, refers to the disclosure of U.S. Pat. No. 3,970,518 and states that there is no literature verification that uncoated magnetic particles may effectively be made to bind directly with antibody. Moreover, the above-noted U.S. Pat. No. 4,554,088 to Whitehead et al., which relates to chemical coupling of antibodies to silanated metal oxide magnetic particles, states that antibodies absorbed on iron oxides are substantially detached by 24 hour incubation at 50.degree. C. in 1M sodium chloride. Furthermore, with respect to superparamagnetic, resuspendable colloidal particles (such as those used in the practice of the present invention), the method of absorption of antibodies proposed in U.S. Pat. Nos. 3,970,518 and 4,018,886 could not easily be made to work on such particles, as the field strength required to capture the particles for washing or subsequent retrieval would be enormous and cannot be achieved with the device proposed. Insofar as is known, apart from the aforementioned colloidal magnetite preparations, there is no currently available way of coating superparamagnetic, resuspendable colloidal particles of iron oxide so as to form a suspensoid, with the dispersed particles having cores of single iron oxide crystals.
As should be apparent from the foregoing, the need for performing bound/free separations is essential to achieving accurate determination of specific target substances. Among the various prior art assays mentioned above, techniques involving second antibody precipitation provide a notable advantage in that reaction of first antibody with labeled and unlabeled analyte proceed with diffusion controlled kinetics. The addition of second antibody reagents or insolubilizing agents in certain instances to cause precipitation, although cumbersome, affords an efficient way of converting soluble macromolecules to an insolubilized state. The introduction of immunological reagents on solid supports results in simplicity of operation, but with markedly slower reaction rates. As an example, coated tube technology requires significantly longer incubation periods and requires constant agitation of the test vessel. Although, mobile solid-phase reagents markedly increase available surface area for reaction and consequently shorten reaction time, such reagents also typically require agitation to maintain a uniform suspension.
In view of the decided advantages afforded by magnetic separations in the assays described above, which include ease of separation, safety (eliminating possible container breakage during centrifugation) and the avoidance of energy-consuming devices to serve as magnetic field sources, a reagent capable of facilitating bound-free separation that normally remains in solution, reacts with the reaction kinetics of macromolecules or colloids and is readily agglomerable would be ideal, particularly if such material were magnetic or could be rendered magnetic, so as to be more easily removable from solution.